Published OnlineFirst April 8, 2015; DOI: 10.1158/0008-5472.CAN-14-2493
Cancer
Research
Priority Report
Tumor-Induced Pressure in the Bone
Microenvironment Causes Osteocytes to Promote
the Growth of Prostate Cancer Bone Metastases
Joseph L. Sottnik1, Jinlu Dai1, Honglai Zhang2, Brittany Campbell1, and Evan T. Keller1,2
Abstract
Cross-talk between tumor cells and their microenvironment
is critical for malignant progression. Cross-talk mediators,
including soluble factors and direct cell contact, have been
identified, but roles for the interaction of physical forces
between tumor cells and the bone microenvironment have not
been described. Here, we report preclinical evidence that
tumor-generated pressure acts to modify the bone microenvironment to promote the growth of prostate cancer bone metastases. Tumors growing in mouse tibiae increased intraosseous
pressure. Application of pressure to osteocytes, the main
mechanotransducing cells in bone, induced prostate cancer
growth and invasion. Mechanistic investigations revealed that
this process was mediated in part by upregulation of CCL5 and
matrix metalloproteinases in osteocytes. Our results defined
the critical contribution of physical forces to tumor cell growth
in the tumor microenvironment, and they identified osteocytes as a critical mediator in the bone metastatic niche. Cancer
Introduction
bone remodeling. Similarly, tumor growth in bone should induce
physical forces that effect osteocytes. The role of osteocytes has yet
to be established in tumor biology even though osteocytes are the
most abundant cell in bone (2). Accordingly, the goal of this study
was to determine whether tumor growth in bone induces physical
forces and determine whether these forces educate osteocytes to
promote tumor progression.
Prostate cancer is the second most common tumor of men in
the United States with bone being the primary location of metastases. There are few therapeutic options for patients with bone
metastases. Bone is a relatively inelastic tissue, with tumor growth
restricted by mineralized extracellular matrix (ECM). Furthermore, prostate cancer bone metastases are typically osteoblastic,
promoting poorly organized bone formation. The lack of potential space present in bone, and continual expansion of tumor,
creates a paradox in which bone must be degraded or physical
pressure within the tumor will increase resultant of continuous
cellular division. Therefore, we hypothesized that tumor growth in
bone would increase pressure within the bone microenvironment.
Bone is comprised of osteoblasts, cells responsible for bone
production, osteoclasts, cells responsible for bone resorption, and
osteocytes, the cells that coordinate the responses of osteoblasts
and osteoclasts. Osteocytes are terminally differentiated osteoblast lineage cells embedded in mineralized bone (1). The primary role of osteocytes is maintaining bone homeostasis by
translating physical forces into biochemical signals, a process
called mechanotransduction (2). Alteration of physical forces in
bone modulates osteocytes biochemical responses regulating
1
Department of Urology, University of Michigan, Ann Arbor, Michigan.
Department of Pathology, University of Michigan, Ann Arbor,
Michigan.
2
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
Corresponding Authors: Evan T. Keller, University of Michigan School of
Medicine, 5308 Cancer Center, 1500 East Medical Center Drive, Ann Arbor, MI
48109-0940. Phone: 734-615-0280; Fax: 734-936-9220; E-mail:
[email protected]; and Joseph L. Sottnik, [email protected]
doi: 10.1158/0008-5472.CAN-14-2493
2015 American Association for Cancer Research.
Res; 75(11); 1–8. 2015 AACR.
Materials and Methods
Cell lines
The MLO-Y4 murine osteocytes cell line (courtesy of Dr. Lynda
Bonewald; University of Missouri, Kansas City, MO) was maintained as previously described (3). DU145, LNCaP, and PC3
(human prostate cancer); BPH-1 (benign prostatic hyperplasia1); ACE1 (canine prostate cancer; courtesy of Dr. Thomas Rosol,
The Ohio State University, Columbus, OH); and H441 and A549
(human lung carcinoma) cell lines were maintained in RPMI with
10% FBS and 1 penicillin/streptomycin. MDA-MB-231 and
MCF7 were cultured in DMEM-based media. All cells were serially
passaged by trypsinization and maintained at 37 C and 5% CO2
in a humidified atmosphere.
Animals
All animal studies were performed in an American Association
for Laboratory Animal Care-approved facility, with approval of
the University Committee on Use and Care of Animals. Male SCID
mice 8 to 10 weeks of age were used for all experiments. Four mice
per group were used for all in vivo experiments.
In vivo pressure measurement in tumor-bearing mice
Mice were anesthetized with isoflurane and 1 106 tumor cells
were injected in the intramedullary cavity of the tibia. Shamtreated mice were injected with an equivalent volume of PBS. 24
hours after tumor challenge, implantation of a wireless pressure
transmitter was performed similar to previous description (4).
Briefly, a DSI PhysioTel PA-C10 pressure transmitter (Data
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Figure 1.
Prostate cancer growth in bone leads
to elevated intramedullary pressure.
A, SCID mice (n ¼ 4/group) were
6
challenged with 1 10 tumor cells 24
hours before implantation of a wireless
pressure monitoring device as depicted in
the schematic. B, mice challenged with
DU145 showed a significant (P < 0.05)
increase in intramedullary pressure, with
a peak average pressure of 37.84 mmHg
16 days after tumor challenge, compared
with sham-injected mice. C, radiographs
depict weakening of the cortical bone
(arrow) beginning 2 weeks posttumor
challenge, with loss of cortical bone 3
weeks after challenge. Initiation of
cortical thinning is associated with the
drop in ImP observed. D, mice challenged
with ACE1 showed similar changes in
pressure as observed in mice challenged
with DU145. E, radiographs from mouse
challenged with ACE-1 show cortical bone
decay similar to DU145 at 2 weeks.
Together, these data show that prostate
cancer growth in bone leads to significant
increases in intramedullary pressure.
Two-way repeated measures ANOVA
was used for statistical comparisons.
Sciences International) was implanted in the abdomen and
catheter inserted into the intramedullary cavity of the tibia where
tumor was previously implanted (Fig. 1A).
Real-time measurements of intramedullary pressure (ImP) were
collected and analyzed using Dataquest A.R.T. System software (DSI
international). Analysis of measurements was initiated 7 days after
catheter implant to allow for inflammation to subside. Measurements were averaged for each mouse and reported. Radiographs
were obtained weekly using a Faxitron MX-20 at 4 magnification.
Conditioned media
Conditioned medium (CM) was prepared by incubating near
confluent MLO-Y4 cells on collagen-coated plates for 24 hours
with RPMI media containing 0.1% FBS and 1 penicillin/streptomycin. Supernatant was removed and centrifuged at 500 g for
5 minutes. MLO-Y4 cells were counted and CM was normalized
by adjusting to a final concentration of 2 107 cells per 30 mL.
BCA assay was used to measure CM protein concentration.
Application of hydrostatic pressure in vitro
Application of hydrostatic pressure to cells in vitro was
accomplished as previously described (5–7) using modifica-
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tions. Briefly, cells were seeded into Opticell (Thermo Fisher
Scientific) cassettes and allowed to adhere overnight. Pressure
was created by connecting the Opticell to an IV bag filled with
cell culture media using arterial line tubing. Opticells were
clamped between perforated stainless steel sheets secured by
binder clips. Hydrostatic pressure was modulated by adjusting
the height of the IV bag. Pressure was determined using the
following formula: P ¼ rh; where P, hydrostatic pressure; r,
fluid density; and h, the height of the IV bag in relation to the
Opticell.
Viability was assessed by pressurizing cells for 24 hours and
counting live cells by Trypan blue staining. CM was isolated from
MLO-Y4 cells by pressurizing cells for 24 hours in RPMI containing 0.1% FBS at 0, 20, and 40 mmHg. CM was prepared as
described above. BCA assay was used to quantify protein concentration of cells; no significant (P > 0.05) differences were
observed after normalization.
Viability
Prostate cancer cells were plated in 96-well plates at a density of
3,000 cells per well and incubated overnight. Media were aspirated and replaced with CM or control medium (RPMI containing
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Pressure Promotes Prostate Bone Metastases
0.1% FBS) and cells incubated for 24 hours. Viability was assessed
following resazurin (Sigma-Aldrich) incubation. Fluorescence
was determined using a SpectraMax M5 plate reader (Molecular
Devices) with Softmax Pro v5.4 software. Relative viable cell
number was expressed as a percentage of control treated cells.
Migration and invasion
Screening of compounds identified in the reagents section was
performed utilizing Culturex Migration and low BME Invasion
assays (Trevigen). Validation of these results were performed
using commercially available Boyden chamber assays with (invasion) and without (migration) Matrigel according to the manufacturer's directions (Corning). Prostate cancer cells (ACE1 ¼ 1 105; DU145 ¼ 5 104; LNCaP ¼ 5 105; and PC3 ¼ 1 105)
were plated in the top well in 0.1% FBS RPMI. In the bottom well,
control media or CM was added as a chemoattractant. Blocking
or neutralizing compounds were incubated with CM or control
media for 1 hour at room temperature before plating. To recapitulate the protein concentration of CCL5 and MMP2 found in
40 mmHg CM, recombinant respective protein was added to 0
mmHg before plating as a chemoattractant. Cell number from five
random 400 fields were counted, averaged for each membrane,
and reported as the mean SEM.
Cytokine array
Identification of MLO-Y4 secreted mediators was achieved
using a commercially available mouse cytokine array (R&D Systems) according to the manufacturer's instructions. Protein concentration was normalized before performing the assay. Pixel
density of each spot was measured using Photoshop CS3 extended
(Adobe Systems Inc.). Spots were averaged, background subtracted, and average values reported for each cytokine.
Reagents
The following reagents were used in screening effector molecules identified in the cytokine array: Rat anti-mouse CCL5 (R&D
Systems), rat IgG2a isotype control (R&D Systems), recombinant
mouse CCL5 (R&D Systems), recombinant MMP2 (R&D Systems), CCR2 antagonist (Calbiochem), rat anti-mouse CXCL10
(R&D Systems), cFMS Receptor III inhibitor (Calbiochem), and
Batimastat (Calbiochem).
Real-time qPCR
RNA was extracted using TRIzol (Invitrogen), purified by the
RNeasy Mini-Kit (Qiagen), and reverse transcribed using the
SuperScript III Reverse Transcriptase Kit (Invitrogen). Quantitative real-time PCR was performed in triplicate using SYBR Green
qPCR Master Mix (Qiagen) in a 10 mL reaction volume on a Roche
LightCycler 480 (Roche). Primers were purchased from SABiosciences (Qiagen). Measurements from triplicate Ct values were
normalized to GAPDH, averaged, and reported.
ELISA
Commercially available ELISA for murine CCL5/RANTES
(R&D Systems) and MMP2 (R&D Systems) were used according
to the manufacturer's instructions.
Zymography
Zymography was performed using CM from osteocytes and
prostate cancer cell lines normalized by total protein as measured
by BCA assay. Ten percent gelatin zymogram gels (Life Techno-
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logies) were used according to the manufacturer's directions and
counterstained using SimplyBlue SafeStain (Life Technologies).
Washed gels were scanned and densitometry performed using
ImageJ (8).
Statistical analysis
All experiments were repeated independently two to three
times. Statistical analysis was performed using Prism 5 (GraphPad
Software). In vivo pressure experiments were analyzed using twoway repeated measures ANOVA with a Bonferroni posttest. Multiple groups were compared using one-way ANOVA with Bonferroni posttest. Data comprising only two groups were analyzed by
a t test. Zymography and neutralization or pressure CM with antiCCL5 antibody were compared using two-way ANOVA with the
Bonferroni posttest. For all statistical analyses, a cutoff of P < 0.05
was used to assess statistical significance.
Results
Tumor growth in bone increases ImP
The primary site of prostate cancer metastasis is bone, in which
tumor growth may induce pressure due to the lack of expansible
space due to mineralized ECM. Therefore, we sought to determine
whether prostate cancer growing in bone significantly altered ImP.
Prostate cancer cells were injected into the tibiae of SCID mice,
which were subsequently cannulated with a wireless pressure
transmitter (Fig. 1A) allowing for real-time monitoring of ImP.
DU145 and ACE1 tumor growth significantly increased ImP,
peaking approximately 15 to 16 days posttumor challenge (Fig.
1B and D). Approximately 21 days posttumor injection, ImP
declined; albeit was still increased compared with baseline.
Radiographic loss of cortical bone was observed at the same time
period of declining ImP (Fig. 1C and E). This observation suggests
that cortical bone loss may mitigate tumor-induced pressure.
Challenge with the highly osteolytic prostate cancer cell line PC3
did not induce a significant increase in ImP (Supplementary Fig.
S1). From these data, it was determined that mice have a basal ImP
of 19.12 8.18 mmHg; similar to previous reports in mice
without tumors (4). An average peak ImP of 38.51 8.63 mmHg
was observed in ACE1 and DU145 across all experiments. Investigation of the role of tumor-generated pressures on prostate
cancer using the values identified from the in vivo studies was
then investigated in vitro.
Effects of increased ImP on osteocytes and prostate cancer
To investigate the impact of increased pressure on osteocytes
and prostate cancer viability in vitro, we applied hydrostatic
pressure, based upon our in vivo findings, to cultures of osteocytes
and prostate cancer cells. Pressure increased MLO-Y4 osteocytes
viability (Fig. 2A). Even though osteocytes are not thought to
proliferate in vivo, this observation speaks to the ability of osteocytes to survive stressful conditions (1, 2). In contrast, pressure
either had no effect (DU145) or decreased prostate cancer (LNCaP
and PC3) cell viability (Fig. 2A).
Osteocytes coordinate bone remodeling by releasing biochemical mediators. Thus, CM was collected from osteocytes and
found to induce prostate cancer viability (Supplementary Fig.
S2). Osteocyte CM also increased cell viability of breast and lung
cancer cell lines, suggesting that the effect is not unique to prostate
cancer (Supplementary Fig. S3). Osteocyte CM induced prostate
cancer migration and invasion when used as a chemoattractant
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Figure 2.
Application of hydrostatic pressure to osteocytes promotes prostate cancer aggressiveness. A, hydrostatic pressure was applied in vitro to MLO-Y4 osteocytes
and DU145, LNCaP, and PC3 prostate cancer cell lines. After 24 hours, cells were counted using Trypan blue staining and live cell count depicted. B, CM was
isolated from MLO-Y4 osteocytes under pressure at 0, 20, and 40 mmHg for 24 hours. Control media (RPMI with 0.1% FBS) was used as a negative control.
Osteocyte CM was then applied to prostate cancer cell lines for 24 hours to determine changes in viability as measured by resazurin. CM was used as a
chemoattractant in Boyden chamber assays to determine alterations in migration and invasion. One-way ANOVA with Bonferroni posttest was used for all analyses;
bars, significant differences of P < 0.05.
(Supplementary Fig. S2). To determine whether increased pressure altered the protumorigenic phenotype conferred by osteocytes, CM was collected from MLO-Y4 cells pressurized at 0
(normal culture conditions), 20 (physiologic baseline ImP), and
40 mmHg (peak pressure observed). CM from pressurized cells
dose dependently increased prostate cancer viability, migration,
and invasion (Fig. 2B). These data provide evidence that tumorinduced pressure promotes secretion of factors associated with
prostate cancer survival and motility in response to physical forces
induced by tumor growth.
Identification and neutralization of CM-derived CCL5
To identify candidate mediators of prostate cancer protumorigenic activity, the cytokine profile of 0 mmHg osteocyte CM was
compared with CM produced at 40 mmHg pressure. Increased
pressure induced numerous chemokines and cytokines (Fig. 3A;
detailed in Supplementary Table S1). Several of the factors with
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altered expression were screened for function on prostate cancer
using neutralizing antibodies and receptor antagonists to inhibit
prostate cancer viability, migration, and invasion. Inhibition of
CCL2, MCSF, and CXCL10 was found to have no effect (data not
shown). CCL5-neutralizing antibody inhibited osteocyte CMinduced prostate cancer migration and invasion. Therefore, CCL5
mRNA was measured by qPCR (Fig. 3B), and ELISA to measure
CCL5 protein concentration in CM (Fig. 3C and Supplementary
Fig. S4A). Pressure induced a dose-dependent increase in CCL5
production from MLO-Y4 cells.
CCL5 is a chemoattractant for T cells that has been implicated in
promoting prostate cancer migration and viability (9, 10). To
further characterize the tumor-promoting role of CCL5, prostate
cancer cells were incubated with recombinant CCL5 (rCCL5).
Although rCCL5 promoted DU145 migration and invasion, it
promoted only migration, and not invasion of LNCaP and PC3
cells (Fig. 3D). Because these cells lines were tested under similar
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Pressure Promotes Prostate Bone Metastases
Figure 3.
Identification of CCL5/RANTES as an osteocyte-secreted mediator of enhanced prostate cancer invasion. A, CM prepared from MLO-Y4 cells pressurized at 0 and 40
mmHg for 24 hours were compared using a cytokine screening antibody array and subjected to densitometry. Results are shown as change in optical
density of the cells subjected to 40 mmHg relative to the 0 mmHg. B, real-time PCR of CCL5 from pressurized MLO-Y4 cells was performed and normalized to
GAPDH. C, CCL5 secreted from osteocytes, normalized by protein concentration, increases as the level of hydrostatic pressure increases as measured by
ELISA. D, recombinant murine CCL5 (rCCL5; 10 mg/mL) was used as a chemoattractant for prostate cancer migration and invasion. Neutralization of rCCL5 was
accomplished by incubating with anti–CCL5-neutralizing antibody (1 mg/mL) but not with isotype control antibody (1 mg/mL). rCCL5 neutralization inhibited
migration across all cell lines. However, invasion of LNCaP and PC3 was not altered by the neutralization of rCCL5. E, CM derived from MLO-Y4 cells pressurized at 0,
20, and 40 mmHg was incubated with CCL5-neutralizing antibody before being used as a chemoattractant in migration and invasion assays. Neutralization
of CM-derived CCL5 led to significant inhibition of prostate cancer migration and invasion despite increasing pressure. Secretion of CCL5 by MLO-Y4 was induced
by tumor-generated pressure, leading to subsequent increases in tumor cell invasiveness. One-way ANOVA with Bonferroni posttest (C and D) or two-way
ANOVA (E) was performed; bars, significant differences where P < 0.05.
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conditions it is likely there may be cell specificity to rCCL5;
however, it is possible that even with the replicate experiments,
there was insufficient statistical power to detect the invasion in
LNCaP and PC3 cells. Neutralization of rCCL5 using an antiCCL5 antibody negated the promigratory effects conferred by
rCCL5 (Fig. 3D). The observation that invasion was not affected
by increased CCL5 alone suggests that osteocyte-produced CCL5
is insufficient to promote invasion by itself.
To investigate whether CCL5 in the context of osteocyte CM
promotes prostate cancer migration and invasion, we evaluated
neutralization of osteocyte-derived CCL5. CCL5 neutralization
inhibited migration in nonpressurized CM (Supplementary Fig.
S4B). Furthermore, neutralization of CM derived from pressurized osteocytes inhibited migration and invasion at all pressures
examined (Fig. 3E). However, inhibition of CCL5 did not
completely block the pressure-induced migration and invasion,
indicating that other factors contribute to this activity. To determine whether CCL5 was sufficient to promote the pressureinduced migration and invasion, we recapitulated the concentration of CCL5 found in 40 mmHg CM (3 ng/mL) by adding rCCL5
to 0 mmHg CM. The addition of CCL5 promoted migration and
invasion to levels similar to 40 mmHg CM (Supplementary Fig.
S4C). These invasion results contrast with the studies using rCCL5
alone (Fig. 3D), suggesting that other factors produced by osteocytes contribute to promoting invasion of prostate cancer in
addition to CCL5.
Neutralization of CM-derived CCL5 and MMP mitigates
prostate cancer proinvasive phenotype
Matrix metalloproteinases (MMP) are known to be expressed
by osteocytes to modulate ECM during bone-resorptive processes
(11). To determine whether osteocytes modulate prostate cancer
invasion through MMPs, CM from MLO-Y4 and prostate cancer
cell lines was isolated and compared using zymography. MLO-Y4
CM produced up to 67 more MMP2 than prostate cancer cell
lines (Supplementary Fig. S5A); MMP9 expression was elevated to
a lesser degree. Pressurization of MLO-Y4 increased secretion of
MMP2 and MMP9 as measured by zymography (BPH-1 was used
as a positive control; Fig. 4A; ref. 12). Quantification of latent
versus active MMPs showed increased MMP2 activity associated
with increased pressure (Fig. 4A and Supplementary Fig. S5B). The
effect of pressure on MMP2 and MMP9 mRNA expression in
osteocytes was evaluated demonstrating pressure induced a dosedependent increase of both MMP2 and MMP9 mRNA (Supplementary Fig. S5C). MMP2 quantitation by ELISA showed
increased protein expression associated with pressure (Fig. 4A).
To determine whether pressure promotes invasion through MMP
production by osteocytes, we added a broad spectrum MMP
inhibitor, batimastat to CM. Batimastat inhibited invasion but
not migration of prostate cancer cells in both nonpressurized CM
(Supplementary Fig. S5D) and 40 mmHg CM (Fig. 4B) cultures,
indicating that in addition to basal expression of MMP's by
osteocytes, pressure-induced MMP expression by osteocytes
increases invasion. Furthermore, addition of recombinant MMP2
(rMMP2) to 0 mmHg CM, to recapitulate the concentration of
MMP2 found in 40 mmHg CM, promoted invasion to levels
similar to 40 mmHg CM alone (Supplementary Fig. S5E), indicating the ability of MMPs to contribute to the pressure-induced
invasion. Our finding that pressure induced CCL5 (Fig. 3B and C)
along with previous reports, indicating that CCL5 promotes MMP
expression led us to explore whether CCL5 promotes MMP
OF6 Cancer Res; 75(11) June 1, 2015
expression in osteocytes (13, 14). Accordingly, rCCL5 was added
to MLO-Y4 and found to increase MMP2 and MMP9 mRNA
expression (Supplementary Fig. S6), suggesting that pressure
could induce MMP2 and MMP9, in part, through CCL5.
Discussion
The consequences of tumor-generated physical forces on the
tumor microenvironment, in general, and the bone microenvironment specifically, have yet to be elucidated. Our data provide
evidence that tumor growth–induced pressure promotes a pro–
proliferative and proinvasive response from osteocytes. It achieves
this, in part, through pressure-induced osteocytes production of
CCL5 and MMPs (model in Fig. 4C). This is the first demonstration of tumor modulating the microenvironment through a
physical force so that the microenvironment enhances cancer
progression.
Herein, data are presented showing that tumors that produce
radiographically mixed lesions (i.e., ACE1 and DU145 cells)
increase ImP. However, no elevation of ImP was observed from
lytic PC3 cells, suggesting that osteolysis provided sufficient space
for PC3 growth without inducing changes in ImP. Consistent with
this possibility is the observation that breast cancer cells respond
to matrix rigidity through upregulation of MMPs (15). It is
plausible that matrix rigidity, through its resistance to conformational changes, would influence the magnitude of pressure generated by a growing tumor. Furthermore, in tumors that create
mixed to osteoblastic lesions, the decreased ability to resorb bone
and subsequent inability to create space for tumor growth may
result in the increased ImP.
The primary role of osteocytes is coordination of bone production and resorption in response to physical stimuli (1, 2). Our
report is the first to demonstrate that osteocytes support cancer
progression. Bone is a complex tissue, and even though osteocytes
are the primary mechanotransducing cells, it is plausible that
other cells may be affected by altered ImP. We observed numerous
cytokines produced from osteocytes in response to hydrostatic
pressure. CCL5 was observed to have a significant response on
promoting tumor invasion and migration. CCL5/RANTES is a
chemokine whose primary role has been defined as a chemoattractant for immune cells. Prostate cancer cells DU145, LNCaP,
and PC3 have previously been shown to express multiple receptors for CCL5 (10). CCL5 has previously been shown to promote
migration of numerous tumor types, including prostate, breast,
lung, and osteosarcoma (9, 10, 16–18). Our results are consistent
with CCL50 s known activity on invasion and migration; however,
as indicated in Supplementary Table S1 and the observation
that CCL5 only partially inhibited of migration and invasion
(Fig. 3E), multiple other factors most likely also contribute to the
pressure-induced migration and invasion.
In addition to CCL5, osteocyte MMP2 and MMP9 basal expression was higher than that of prostate cancer cell lines and further
induced by pressure. Batimastat inhibited invasion of prostate
cancer toward osteocyte-derived CM, indicating a role for MMPs
in osteocyte-mediated invasion. Although MMPs are known
to play a role in cancer invasion their expression from osteocytes
as potentiating tumor invasion is a novel finding (19). Osteocytes
have been observed to have some bone resorption properties,
and MMP expression is likely a consequence of this function (11)
and MMP2 is important in osteocyte canalicular network formation and maintenance (20). Osteocyte-derived MMPs provide
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Figure 4.
Inhibition of osteocyte-derived
MMPs mitigates protumorigenic
responses. A, MLO-Y4 was subjected
to 0, 20, or 40 mmHg pressure for
24 hours; CM was prepared and
normalized by protein concentration
as measured by BCA assay before
analysis by zymography. BPH-1 was
used as a positive control for MMP
expression and plain cell culture
media used as a negative control.
Bands were subjected to
densitometry and reported as a
percentage of active MMP2 and
MMP9. CM was normalized by protein
concentration, as measured by BCA,
and MMP2 analyzed by ELISA. B, MLOY4 CM was incubated with batimastat,
a broad spectrum MMP inhibitor, to
inhibit osteocyte-derived MMP. Forty
mmHg CM with batimastat or DMSO
control was then used to determine
alterations in prostate cancer
migration and invasion. C, a proposed
model system for tumor-induced
pressure promoting prostate cancer
aggressiveness. Prostate cancer
proliferation promotes pressure in
bone, leading to induction of
osteocyte production of CCL5, which
can induce MMPs, and MMPs
themselves. MMPs degrade the bone
matrix releasing a variety of growth
factors, whereas the combination of
CCL5 and MMPs promote further
invasion into bone. One-way ANOVA
with Bonferroni posttest used for
ELISA analysis; bars, significant
differences of P < 0.05. The Student
two-tailed t test was used to analyze
MMP inhibition experiments. All bars
represent significant differences
where P < 0.05.
a mechanism for nutrient release and development of a vicious
cycle previously associated with tumor growth in bone (21, 22).
Similarly in prostate cancer, integrins, such as avb6, may interact
with TGFb, leading to MMP2 induction and osteolytic degradation (12). This is especially interesting in the context that MMP
activation of TGFb is important in osteoblast viability and osteocyte differentiation (23). Furthermore, our results showing that
CCL5 induces MMPs in osteocytes supports previous findings
where CCL5 promoted MMP production in tumor cells (13, 14)
and provides a potential mechanism to account for pressureinduced MMP expression. In this setting, tumor promotion of
bone degradation may elicit growth factor release from mineralized bone, further promoting tumor growth through initiation of
a positive feedback loop. The response of osteocytes to tumorgenerated pressure has great potential to promote the vicious cycle
as osteocytes are the most abundant cell in bone and have a greater
surface area than that of trabeculae (24).
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Conclusion
The current report demonstrates that tumor growth promotes
ImP and these physical forces promote osteocyte secretion of the
tumor-promoting factors CCL5 and MMPs. Furthermore, osteocytes were identified as novel protumorigenic cellular mediators.
Exploration of osteocytes and their response to physical forces is
necessary to develop novel therapeutic targets for the inhibition of
bone metastases.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: J.L. Sottnik, E.T. Keller
Development of methodology: J.L. Sottnik, J. Dai, H. Zhang, E.T. Keller
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): J.L. Sottnik, J. Dai, B. Campbell
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Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): J.L. Sottnik, J. Dai, B. Campbell, E.T. Keller
Writing, review, and/or revision of the manuscript: J.L. Sottnik, J. Dai, E.T. Keller
Administrative, technical, or material support (i.e., reporting or organizing
data, constructing databases): J.L. Sottnik
Study supervision: E.T. Keller
using the DSI system and pressure measurement. The authors thank Dr. Gene
DiResta at the NYU Polytechnic School of Engineering and Dr. John Healey at
Memorial Sloan Kettering Cancer Center for their help with the in vitro pressure
system.
Grant Support
Acknowledgments
The authors thank Dr. Lynda Bonewald at the University of Missouri-Kansas
City for providing the MLO-Y4 cell line. The authors thank Dr. John Frangos and
Diana Meays of the La Jolla Bioengineering Institute for their instruction
concerning the technique to measure ImP in mice. The authors thank Dr. Louis
D'Alecy and Steven Whitesall at the University of Michigan for their expertise in
This work was supported by a Department of Defense Prostate Cancer
Research Program Training Award (W81XWH-12-1-0172; J.L. Sottnik) and
NIH grants R01CA190554, P01CA093900, and S10RR026405.
Received September 5, 2014; revised March 24, 2015; accepted March 25,
2015; published OnlineFirst April 8, 2015.
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Cancer Research
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 2015 American Association for Cancer
Research.
Published OnlineFirst April 8, 2015; DOI: 10.1158/0008-5472.CAN-14-2493
Tumor-Induced Pressure in the Bone Microenvironment
Causes Osteocytes to Promote the Growth of Prostate
Cancer Bone Metastases
Joseph L. Sottnik, Jinlu Dai, Honglai Zhang, et al.
Cancer Res Published OnlineFirst April 8, 2015.
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